Splicing is the maturation process for nuclear pre-mRNAs to have their noncoding intron sequences removed and their exons linked to form functional mRNAs. Many pre-mRNAs are also alteratively spliced to produce mRNAs of different functions. Thus, splice site selection and the splicing reaction not only have to be precise, but also can be regulated. The long term goal of the proposed research is to understand the mechanism of pre-mRNA splicing and regulation of gene expression at the RNA processing level in mammalian cells. Our interest will focus on the role and regulation of the human splicing factor SC35. SC35 is required for spliceosome assembly and for the first step of the splicing reaction. We have cloned the cDNA that encodes SC35. Recombinant SC35 produced in a baculovirus system can complement the nuclear extracts in which SC35 has been removed by immunodepletion using a specific anti-SC35 monoclonal antibody. In specific Aim 1, this in vitro splicing-complementation system will be used to study the function of SC35 from two angles: the interaction between SC35 and pre-mRNA and the interaction between SC35 and other spliceosomal components. The biological consequence of these interactions in splicing and in splice site selection will then be determined. These studies may lead to the identification of additional splicing factors. Based on the observations that SC35 effects splice site selection in vitro, Aim 2 will investigate the role of SC35 in regulation of alternative splicing. Aim 3 will address regulation of the SC35 activity as a means to regulate splicing. Specifically, we will study phosphorylation of SC35 and its biological significance. These studies will be extended to the identification of protein kinases involved in modifying SC35 and have a potential to make connections to various signal transduction pathways. Aim 3 will also include studying regulation of SC35 at the RNA level by investigating the possibility that the splicing factor SC35 may have isoforms as a result of alternative splicing. Finally in Aim 4, additional monoclonal antibodies against mammalian spliceosomal components will be further characterized as a part of our long term effort in understanding the mechanism of splicing by studying the functions of splicing factors involved in various aspects of the splicing reaction. Initially, the experimental strategies developed for SC35 will be used to characterize these splicing factors. Overall, the results of the proposed biochemical studies of the human splicing factor SC35 should provide further sights into the mechanism of splicing, splice site selection, and regulation of gene expression at the RNA processing level.